The formation of multicellular communities requires extensive intracellular communication and although much has been learned about the intricate regulatory network involved in differentiation and biofilm formation, relatively little is known about the signals responsible for initiating bacterial multicellularity. Recently, a novel mechanism for triggering multicellularity in Bacillus subtilis was described and surprisingly, the signaling mechanism involves a histidine kinase activated by potassium leakage. In response to potassium leakage caused by natural products, such as nystatin and surfactin, the histidine kinase KinC initiates a signal transduction cascade that culminates in biofilm formation. Discovery of the novel signaling mechanism provides an opportunity to perform a rigorous functional analysis of the potassium sensing histidine kinase and to determine whether and how other genes are involved in sensing potassium leakage. I propose to address three specific aims: I) evaluate the role of polycyclic terpenoids in signal sensing, II) assess the role of potassium uptake in this signal transduction mechanism, and III) identify key KinC residues involved in signal sensing and determine if there is a cofactor involved. The proposed research is designed to both lay the foundation for future studies of protein kinases capable of sensing changes in intracellular ion concentration and allow me to become proficient using advanced genetic and molecular techniques in a variety of bacterial backgrounds. Studies of this new signaling mechanism will help advance our understanding of large sensory systems and will also have important implications in other research fields, including cancer research and infectious disease research. PUBLIC HEALTH RELEVANCE: Many bacterial and mammalian histidine kinases are homologous and due to structural and functional similarities, it is possible to study model bacterial systems and gain important insight into mammalian systems. By increasing our understanding of the molecular basis for histidine kinase activity in bacteria, it may be possible to design more effective strategies to control or modulate defective mammalian protein kinases that cause cells to become cancerous and/or develop more effective ways to fight infections caused by bacterial biofilms.